Summary
Inhibition of neurotransmitter release by tetanus toxin and botulinum neurotoxin A can be mimicked by intracellular application of the corresponding toxin light chains. The aim of this study was to determine whether the two-chain toxins are reduced by brain preparations to yield free light chains which would represent the ultimate toxins.
The interchain disulfide of two-chain tetanus toxin was cleaved by rat cortex homogenate fortified with NADPH. Reduction was promoted further by addition of thioredoxin. Thioredoxin reductase was demonstrated in and purified from porcine brain cortex. The thioredoxin system which consisted of purified enzyme, thioredoxin and NADPH reduced both toxins. The resulting light chains appeared homogeneous in SDS gel electrophoresis. The complementary heavy chain of tetanus but not of botulinum toxin migrated in two bands, the faster one with the velocity of heavy chain obtained by chemical reduction. The major, slower form was converted into the faster by chemical but not by enzymatic reduction. Tetanus toxin, whether in its single-chain or two-chain version also occurred in two forms which differed by their electrophoretic mobility. The two forms of single-chain toxin were interconverted by chemical reduction or oxidation but not by the thioredoxin system.
It is concluded that a) a thioredoxin system in brain tissue reduces the interchain disulfide of two-chain tetanus toxin and botulinum neurotoxin A, b) tetanus toxin but not botulinum neurotoxin A consists of two electrophoretically distinct forms which differ by the thiol-disulfide status of their heavy chains, c) the disulfide loop within the heavy chain of tetanus toxin is resistant to the thioredoxin system.
Similar content being viewed by others
References
Ahnert-Hilger B, Bizzini B, Goretzki K, Müller H, Völckers C, Habermann E (1983) Monoclonal antibodies against tetanus toxin and toxoid. Med Microbiol Immunol 172: 123–135
Ahnert-Hilger G, Weller U, Dauzenroth ME, Habermann E, Gratzl M (1989) The tetanus toxin light chain inhibits exocytosis. FEBS Lett 242: 245–248
Barbieri L, Battelli MG, Stirpe F (1982) Reduction of ricin and other plant toxins by thiol: protein oxidoreductases. Arch Biochem Biophys 216: 380–383
Bernstein HG, Ansorge S, Reiser M, Müller M, Dorn A (1986) Postnatal changes in thiol: protein-disulfide oxidoreductase immunoreactivity of rat brain. Acta Histoch [Suppl] 33: 273–276
Bernstein HG, Ansorge S, Novakova V, Reiser M, Dorn A (1988) Immunohistochemical analysis of thiol: protein disulfide oxidoreductase in hypothalamic neurons of brattleboro rat. Endocrinol Experiment 22: 41–44
Binz T, Kurazono H, Wille M, Frevert J, Wernars K, Niemann H (1990) The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins. J Biol Chem 265: 9153–9158
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254
Burnette WN (1981) ‘Western blotting’: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195–203
DasGupta B (1989) The structure of botulinum neurotoxin. In: Simpson LL (ed) Botulinum neurotoxin and tetanus toxin. Academic Press, San Diego, pp 53–67
de Paiva A, Dolly O (1990) Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes. FEBS Lett 277: 171–174
Edman JC, Ellis L, Blacher RW, Roth RA, Rutter WJ (1985) Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature 317: 267–270
Eisel U, Jarausch W, Goretzki K, Henschen A, Engels J, Weller U, Hudel M, Habermann E, Niemann H (1986) Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins. EMBO J: 2495–2502
Fedinec AA, Schweitzer JB, Lazarovici P, Yavin E, Bizzini B (1989) Two tetanus toxins with different affinities for polysialogangliosides. 2. Retrograde transport in sciatic nerves to the spinal cord. In: Nistico G et al (eds) Eighth Internatl Conf Tetanus. Pythagoras, Rome, pp 127–134
Freedman RB (1984) Native disulphide bond formation in protein biosynthesis: evidence for the role of protein disulphide isomerase. Trends Biochem Sci 9: 438–441
Goretzki K, Habermann E (1985) Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Med Microbiol Immunol 174: 139–150
Habermann E, Weller U, Hudel M (1991) Limited proteolysis of single-chain tetanus toxin by tissue enzymes, in cultured brain tissue and during retrograde axonal transport to the spinal cord. Naunyn-Schmiedeberg's Arch Pharmacol 343: 323–329
Hawkins HC, De Nardi M, Freedman RB (1991a) Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian disulphide-isomerase. Biochem J 275: 341–348
Hawkins HC, Blackburn EC, Freedman RB (1991b) Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalyzing disulphide isomerization in a protein substrate. Biochem J 275: 349–353
Hillson DA, Lambert N, Freedman RB (1984) Formation and isomerization of disulfide bonds in proteins: Protein disulfide-isomerase. Methods Enzymol 107: 281–294
Holmgren A (1977) Bovine thioredoxin system. Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction. J Biol Chem 252: 4600–4606
Holmgren A (1979) Reduction of disulfides by thioredoxin. Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action. J Biol Chem 254: 9113–9119
Holmgren A (1985) Thioredoxin. Annu Rev Biochem 54: 237–271
Holmgren A (1989) Thioredoxin and glutaredoxin systems. J Biol Chem 264: 13 963–13 966
Kosower NS, Kosower EM, Wertheim B, Correa WS (1969) Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem Biophys Res Commun 37: 593–596
Krieglstein K, Henschen A, Weller U, Habermann E (1990) Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin. Eur J Biochem 188: 39–45
Lazarovici P, Tayot JL, Yavin E (1984) Affinity chromatographic purification and characterization of two iodinated tetanus toxin fractions exhibiting different binding properties. Toxicon 22: 401–413
Lazarovici P, Yanai P, Yavin E (1987) Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution. J Biol Chem 262: 2645–2651
Lundström J, Holmgren A (1990) Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity. J Biol Chem 265: 9114–9120
Luthman M, Holmgren A (1982) Rat liver thioredoxin and thioredoxin reductase: Purification and characterization. Biochemistry 21: 6628–6633
Matsuda M (1989) The structure of tetanus toxin. In: Simpson LL (ed) Botulinum neurotoxin and tetanus toxin. Academic Press, San Diego, pp 69–92
Mochida S, Poulain B, Weller U, Habermann E, Tauc L (1989) Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain. FEBS Lett 253: 47–51
Moss J, Stanley SJ, Morin JE, Dixon JE (1980) Activation of choleragen by thiol: protein disulfide oxidoreductase. J Biol Chem 255:11085–11087
Parton RG, Davison MD, Critchley DR (1989) Comparison of the binding characteristics of two different preparations of tetanus toxin to rat brain membranes. Toxicon 27:127–135
Pigiet VP, Schuster BJ (1986) Thioredoxin-catalysed refolding of disulfide-containing proteins. Proc Natl Acad Sci USA 83: 7643–7647
Poulain B, Tauc L, Maisey EH, Wadsworth JDF, Mohan M, Dolly JO (1988) Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain. Proc Natl Acad Sci USA 85:4090–4093
Rozell B, Hansson HA, Luthman M, Holmgren A (1985) Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats. Eur J Cell Biol 38:79–86
Schiavo G, Papini E, Gerna G, Montecucco C (1990) An intact interchain disulfide is required for the neurotoxicity of tetanus toxin. Infect Immun 58:4136–4141
Stecher B, Weller U, Habermann E, Gratzl M, Ahnert-Hilger G (1989) The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells. FEBS Lett 255:391–394
Stemme S, Hansson HA, Holmgren A, Rozell B (1985) Axoplasmic transport of thioredoxin and thioredoxin reductase in rat sciatic nerve. Brain Res 359:140–146
Weller U, Mauler F, Habermann E (1988) Tetanus toxin: Biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis. Naunyn-Schmiedeberg's Arch Pharmacol 338:99–106
Weller U, Dauzenroth ME, Meyer zu Heringdorf D, Habermann E (1989) Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties. Eur J Biochem 182:649–656
Author information
Authors and Affiliations
Additional information
Send offprint requests to E. Habermann at the above address
Rights and permissions
About this article
Cite this article
Kistner, A., Habermann, E. Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain. Naunyn-Schmiedeberg's Arch Pharmacol 345, 227–234 (1992). https://doi.org/10.1007/BF00165741
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00165741